1. Field of the Invention
The present invention relates to methods of enhancing hybridoma fusion efficiencies through cell synchronization of the fusion partners, in order to aid in production of antibodies.
2. Related Art
The use of monoclonal antibodies (mAbs) as therapeutic reagent has become an effective approach for the treatment of various diseases. In addition, mAbs are powerful tools to gain a better understanding of the immuno-pathogenesis of various diseases.
Generation of monoclonal antibodies has first been described by Kohler and Milstein (Kohler and Milstein, Nature 256: 495-497 (1975)) using the hybridoma technology and since then has become a standard procedure in the lab. A typical protocol for hybridoma generation involves: (i) immunizing an animal (e.g., mouse, rat or rabbit) with a purified protein antigen; (ii) harvesting antibody producing B-cells, typically from the spleen; (iii) fusing B-cells with a non-secretory myeloma cell line deficient for the enzyme hypoxanthine guanine phosphoribosyl transferase (e.g., x63-Ag 8. 653 from a BALB/c mouse strain); (iv) growing hybridoma cells in a selection medium containing hypoxanthine, aminopterin and thymidine (HAT) and (v) screening for cells that produce the desired antibody and (vi) limit dilution cloning to obtain a homogenous cell line that secretes the antibody.
In this time consuming multi-step process, one of the most critical steps is cell fusion, where antibody-secreting B cells and myeloma cells are fused. The murine HGPRT negative B cell myeloma cell line, FO, with a doubling time of 8-12 hours, has become a standard fusion partner in the hybridoma process. When fused to splenocytes from a mouse that is mounting a strong humoral immune response, FO-derived hybridomas that secrete the mAb of interest can be generated quickly.
However, the cell fusion process is very inefficient. Even under best known conditions, such as purity of reagents, temperature, and cell viability, only approximately 0.08% splenic antibody secreting cells are fused. Such inefficiency is due to many factors. For example, fusion of two splenic cells creates a hybridoma that is not immortalized and will not grow, and fusion of two myeloma cells creates a hybridoma that does not secrete antibody and will die under selection media. In addition, fusion of greater than two cells results in unstable syncytias and the polykaryons will not survive.
As a result of all these variables, initial hybridomas are often unstable and do not survive the selection process while the ones that do usually secrete low levels of antibody. Therefore, there is a need for improved methods to generate hybridomas and make monoclonal antibodies.